Enzyme immunoassays and radioimmunoassays have been developed to determine the existence and concentration of antibodies in body fluids or tissues from animals, humans, and plants. The enzyme-linked immunosorbent assay (ELISA) is one type of enzyme immunoassay used as a diagnostic tool for this purpose. Several different ELISA methods are available. The indirect method for antibody assay uses an antigen attached to a support. The antibody is reacted with an antigen specific for the antibody and allowed to bind to the antigen. The resulting antigen-antibody complex is incubated with an enzyme-labeled antiprimary species of immunoglobulin (i.e., cat-dog). An enzyme substrate is then added, and the amount of enzymatic activity is measured. The enzyme activity is related to the amount of antibody bound to the antigen.
These immunoassays are based on the premise that the clinician can obtain a sufficient amount of specimens of the fluids suspected of containing the antibody to enable performance of the test. Typically, the specimen size for such tests has been 150 microliters (.mu.l) or more. In the case of small animals and newborn infants, this size poses a problem in obtaining a sample of body fluid in sufficient quantity to carry out the test.
An object of the present invention is to enable the performance of such a test with only about one .mu.l of the body fluid.
Enzyme immunoassays have been desirable because the direct visualization of an antigen-antibody complex is thereby possible, using a chromogenic indicator. The interpretation of the test result has been dependent on a proper colorimetric determination. The color is not always easily observed with the unaided eye because of the background color in the environment of the specimen under test.
A dot-immunobinding assay for monoclonal and other antibodies has been proposed as a solution to these problems. The principle of that type of assay is as follows: A dilute solution or suspension of antigen is "dotted" on to a white, nitrocellulose piece of filter paper, and the dot is then incubated with test antibody and with a peroxidase-conjugated second antibody directed against the first antibody. After the development of the peroxidase, a positive reaction is detected as a colored dot against the white filter paper background. This assay has two advantages over other immunoassays. First, the amounts of the antigen and antibody needed for the assay are less than was needed before, because of the small spot size. Second, the nitrocellulose paper provides a background which is almost white, so that viewing is easier. See Analytical Biochemistry, 119, 142-147 (1982).
A major disadvantage of the dot-immunobinding assay is that it involves a large number of dilution, incubation, and washing steps that are required to carry out the test, and these steps consume considerable elapsed time. As many as twenty-one steps are required in such an assay, and each step is time-consuming and subject to error. A standard assay of this type takes four to twenty-four hours to perform.
The same problems occur when testing for a suspected antigen.
Accordingly, an improved immunoassay for determining the existence and concentration of an antibody or antigen in a sample is needed. The immunoassay should be operable with extremely small quantities of the fluid being tested; the antigen-antibody reaction should be measured by means of a tagged immunoglobulin, and when the determination is based on a colorimetric determination, the color should be easily observed in the test environment; the assay should require a relatively few number of dilution, incubation, and washing steps, and it should provide accurate results in a relatively short time period.
Achieving these desirable results is among the objects of this invention.